LOCUS       sequence-430879-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The DuET construct encodes the named human GPCR engineered with a 
            cleaved hemagglutinin signal peptide, a 5' FLAG and a 3' 1D4 epitope
            tag optimized for expression in mammalian cells and proteomics 
            studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  AUTHORS   Dahl L, Kotliar IB, Bendes A, Dodig-Crnkovic T, Fromm S, Elofsson A,
            Uhlen M, Sakmar TP, Schwenk JM
  TITLE     Multiplexed selectivity screening of anti-GPCR antibodies.
  JOURNAL   Sci Adv. 2023 May 3;9(18):eadf9297. doi: 10.1126/sciadv.adf9297. 
            Epub 2023 May 3.
  PUBMED    37134173
REFERENCE   2  (bases 1 to 6746)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2023-05-3"; volume: "9(18):eadf9297. doi"; pages: " 
            10.1126/sciadv.adf9297. Epub 2023 May 3"
COMMENT     Sequence Label: OPN3-DuET
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    5..21
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(29..35,36..53,54..59))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    74..95
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(212..229)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(383..971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(463..482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1142..1933,1934..2002))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1765..1784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2003..2107)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2182..2201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2373..2752
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2753..2956
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2907..2927
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3001..3020
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3001..3019
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4475..4492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4481..4705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4751..5179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4838..4857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5048..5069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5188..5208)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5193..5522
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5373..5508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5435..5454
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5589..6383
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5643..5662)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6253..6272
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6557..6678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6594..6613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6648..6667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6727..6743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6727..6743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(join(6740..6746,1..16))
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
       61 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
      121 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
      181 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
      241 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
      301 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      361 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      421 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      481 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      541 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      601 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      661 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      721 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      781 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacggta tttggtatct
      841 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      901 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      961 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     1021 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     1081 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     1141 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     1201 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     1261 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1321 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1381 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1441 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1501 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1561 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1621 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1681 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1741 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1801 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1861 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1921 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1981 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     2041 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     2101 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     2161 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     2221 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     2281 aaggcaaggc ttgaccgaca tttgcatgaa gaatctgctt agggttaggc gttttgcgct
     2341 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
     2401 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2461 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2521 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2581 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2641 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2701 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2761 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2821 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2881 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2941 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     3001 taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttagcgcc
     3061 accatgaaga cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag
     3121 gacgatgatg acgccagcat cgatatgtat agcggtaacc gcagcggagg acacggctat
     3181 tgggatggcg gcggcgccgc tggggccgaa ggtcctgcac ctgctggcac actgtctccc
     3241 gcccctctgt tttctcctgg gacctatgaa aggcttgccc tcctccttgg cagtattggc
     3301 ctcctgggag ttggtaataa tctccttgtc ctggtcctgt actacaagtt ccagagactt
     3361 cggactccga ctcacttgct gctggttaac atatctctga gtgacctcct ggtcagcctg
     3421 tttggcgtca ccttcacttt tgtttcttgt cttaggaatg gttgggtgtg ggacacggtg
     3481 ggctgcgtct gggatggctt tagcggaagt ctcttcggta tagttagtat cgctacactg
     3541 accgtgctgg cttacgagcg atatattcgc gtggttcacg cgagagtgat taactttagt
     3601 tgggcatgga gagctatcac gtatatttgg ctgtacagtc tggcttgggc gggggctccc
     3661 ttgctcgggt ggaatcggta tatactcgac gtgcacggtt tgggatgtac ggttgactgg
     3721 aaatctaagg atgctaacga tagtagcttc gtattgttcc tgtttcttgg gtgtctcgtt
     3781 gtgccgctgg gcgtgatcgc tcactgttat ggacatattc tctactcaat taggatgctg
     3841 cggtgcgtgg aggatctcca aacgattcag gtgatcaaga tcctcaaata cgaaaagaaa
     3901 ctggctaaga tgtgtttcct gatgatcttc acttttcttg tatgctggat gccatatatc
     3961 gttatttgtt ttctggtggt gaacggccac ggtcatctgg tgactccaac tatctccatc
     4021 gtgtcatatc tgttcgcaaa gagtaatact gtttacaacc ccgtcattta tgtctttatg
     4081 atcagaaagt tcaggagaag cctcttgcag ctcctgtgtc tgaggctgtt gaggtgtcag
     4141 cggccagcaa aagacctccc tgcagcaggc tctgagatgc agatcaggcc cattgtcatg
     4201 tcccaaaagg acggagatcg gcctaaaaag aaggtcacat tcaatagttc ctctatcatt
     4261 ttcattatca cctcagatga gtccctgtct gtggacgact ccgacaagac caatggaagc
     4321 aaagtggatg ttattcaggt gcggcctctt atcgataccg gtggacgcac cggtgaattc
     4381 gatgaggcca gcaccaccgt gtctaagacc gagacttctc aggtggcccc tgcctaactc
     4441 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4501 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4561 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4621 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4681 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     4741 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4801 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4861 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4921 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4981 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5041 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5101 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5161 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5221 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5341 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5401 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5461 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5521 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     5581 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5641 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     5701 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5761 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5821 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5881 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5941 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     6001 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     6061 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     6121 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6181 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6241 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6301 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6361 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     6421 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6481 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     6541 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6601 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6661 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     6721 atcatggtca tagctgtttc ctgtgt
//
